Combinatorial strategies for polymer synthesis

ABSTRACT

A method for producing polymer arrays by spacing a dispenser a distance from a surface of a support, dispensing a volume containing a monomer in a single coupling step of less than 5 nl to occupy a localized area of less than 1 cm 2  of the surface of the support, allowing the monomer to bind directly or indirectly to the support and repeating the steps to produce an array of at least 100 polymer ligands at a density of 1000 per cm 2  or greater.

This application is a continuation of U.S. patent application Ser. No.09/498,554, filed Feb. 4, 2000; which in turn is a continuation of U.S.patent application Ser. No. 09/129,463, filed Aug. 4, 1998, now U.S.Pat. No. 6,040,193; which in turn is a continuation of U.S. patentapplication Ser. No. 08/426,202, filed Apr. 21, 1995, now U.S. Pat. No.6.136,269; which in turn is a continuation of U.S. patent applicationSer. No. 07/980,523, filed Nov. 20, 1992, now U.S. Pat. No. 5,677,195;which in turn is a continuation-in-part of U.S. patent application Ser.No. 07/796,243, filed Nov. 22, 1991, now U.S. Pat. No. 5,384,261 andU.S. patent application Ser. No. 07/874,849, filed Apr. 24, 1992, nowU.S. Pat. No. 5,412,087.

BACKGROUND OF THE INVENTION

The present invention relates to the field of polymer synthesis andscreening. More specifically, in one embodiment the invention providesan improved method and system for synthesizing arrays of diverse polymersequences. According to a specific aspect of the invention, a method ofsynthesizing diverse polymer sequences such an peptides oroligonucleotides is provided. The diverse polymer sequences may be used,for example, in screening studies for determination of binding affinity.

Methods of synthesizing desired polymer sequences such as peptidesequences are well known to those of skill in the art. Methods ofsynthesizing oligonucleotides are found in, for example, OligonucleotideSynthesis: A Practical Approach, Gate, ed., IRL Press, Oxford (1984),incorporated herein by reference in its entirety for all purposes. Theso-called “Merrifield” solid phase peptide sysnthesis has been in commonuse for several years and is described in Merrifield, J. Am. Chem. Soc.(1963) 85:2149-2154, incorporated herein by reference for all purposes.Solid-phase synthesis techniques have been provided for the synthesis ofseveral peptide sequences on, for example, a number of “pin.” See e.g.,Geysen et al., J. Immun. Meth. (1987) 102:259-274, incorporated hereinby reference for all purposes. Other solid-phase techniques involve, forexample, synthesis of various peptide sequences on different cellulosedisks supported in a column. See Frank and Doring, Tetrahedron (1988)44:6031-6040, incorporated herein by reference for all purposes. Stillother solid-phase techniques are described in U.S. Pat. No. 4,728,502issued to Hamill and WO 90/00626 (Beattie, inventor).

Each of the above techniques produces only a relatively low densityarray of polymers. For example, the technique described in Geysen et al.is limited to producing 96 different polymers on pins spaced in thedimensions of a standard microtiter plate.

Improved methods of forming large arrays of peptides, oligonucleotides,and other polymer sequences in a short period of time have been devised.Of particular note, Pirrung et al., U.S. Pat. No. 5,143,854 (see alsoPCT Application No. WO 90/15070) and Fodor et al., PCS Publication No. W92/10092, all incorporated herein by reference, disclose methods offorming vast arrays of peptides and other polymer sequences using, forexample, light-directed synthesis techniques. See also, Fodor et al.,Science (1991) 251:767-777, also incorporated herein by reference forall purposes.

Some work has been done to automate synthesis of polymer arrays. Forexample, Southern, PCT Application No. WO 89/10977 describes the use ofa conventional pen plotter to deposit three different monomers at twelvedistinct locations on a substrate. These monomers were subsequentlyreacted to form three different polymers, each twelve monomers inlength. The Southern Application also mentions the possibility of usingan ink-jet printer to deposit monomers on a substrate. Further, in theabove-referenced Fodor et al., PCT application, an elegant method isdescribed for using a computer-controlled system to direct a VLSIPS™procedure. Using this approach, one heterogenous array of polymers isconverted, through simultaneous coupling at a number of reaction sites,into a different heterogenous array. This approach is referred togenerally as a “combinatorial” synthesis.

The VLSIPS™ techniques have met with substantial success. However, insome cases it is desirable to have alternate/additional methods offorming polymer sequences which would not utilize, for example, light asan activator, or which would not utilize light exclusively.

SUMMARY OF THE INVENTION

Methods and devices for synthesizing high-density arrays of diversepolymer sequences such as diverse peptides and oligonucleotides areprovided by virtue of the present invention. In addition, methods anddevices for delivering (and, in some eases, immobilizing) availablelibraries of compounds on specific regions of a substrate are providedby this invention. In preferred embodiments, various monomers or otherreactants are delivered to multiple reaction sites on a single substratewhere they are reacted in parallel.

According to a preferred embodiment of the invention, a series ofchannels, grooves, or spots are formed on or adjacent a substrate.Reagents are selectively flowed through or deposited in the channels,grooves, or spots, forming an array having different compounds—and insome embodiments, classes of compounds—at selected locations on thesubstrate.

According to the first specific aspect of the invention, a block havinga series of channels, such as grooves, on a surface thereof is utilized.The block is placed in contact with a derivatized glass or othersubstrate. In a first step, a pipettor or other delivery system is usedto flow selected reagents to one or more of a series of aperturesconnected to the channels, or place reagents in the channels directly,filling the channels and “striping” the substrate with a first reagent,coupling a first group of monomers thereto. The first group of monomersneed not be homogenous. For example, a monomer A may be placed in afirst group of the channels, a monomer B in a second group of channels,and a monomer C in a third group of channels. The channels may in someembodiments thereafter be provided with additional reagents, providingcoupling of additional maonomers to the first group of monomers. Theblock is then translated or rotated, again placed on the substrate, andthe process is repeated with a second reagent, coupling a second groupof monomers to different regions of the substrate. The process isrepeated until a diverse set of polymers of desired sequence and lengthis formed on the substrate. By virtue of the process, a number ofpolymers having diverse monomer sequences such as peptides oroligonucleotides are formed on the substrate at known locations.

According to the second aspect of the invention, a series ofmicrochannels or microgrooves are formed on a substrate, along with anappropriate array of microvalves. The channels and valves are used toflow selected reagents over a derivatized surface. The microvalves areused to determine which of the channels are opened for any particularcoupling step.

Accordingly, one embodiment of the invention provides a method offorming diverse polymer sequences on a single substrate, the substratecomprising a surface with a plurality of selected regions. The methodincludes the steps of forming a plurality of channels adjacent thesurface, the channels at least partially having a wall thereof definedby a portion of the selected regions; and placing selected reagents inthe channels to synthesize polymer sequences at the portion of theselected regions, the portion of the selected regions comprisingpolymers with a sequence of monomers different from polymers in at leastone other of the selected regions. In alternative embodiments, thechannels or flow paths themselves constitute the selected reactionregions. For example, the substrate may be a series of adjoiningparallel channels, each having reaction sites therein.

According to a third aspect of the invention, a substrate is providedwhich has an array of discrete reaction regions separated from oneanother by inert regions. In one embodiment, a first monomer solution isspotted on a first set of reaction regions of a suitably derivatizedsubstrate. Thereafter, a second monomer solution is spotted on a secondset of regions, a third monomer solution is spotted on a third set andso on, until a number of the regions each have one species of monomerlocated therein. These monomers are reacted with the surface, and thesubstrate is subsequently washed and prepared for reaction with a newset of monomers. Dimers, trimers, and larger polymers of controlledlength and monomer sequence are prepared by repeating the above stepswith different groupings of the reaction regions and monomer solutions.In alternative embodiments, the polymers or other compounds of the arrayare delivered to the regions as complete species, and thus the abovepolymer synthesis steps are unnecessary.

In a preferred embodiment, a plurality of reaction regions on thesubstrate surface are surrounded by a constraining region such as anon-wetting region which hinders the transport of reactants betweenadjacent reaction regions. Thus, the reactants in one region cannot flowto other regions where they could contaminate the reaction. In certainpreferred embodiments, the regions of the array are defined by selectiveirradiation of a substrate surface containing photolabile hydrophobicprotecting groups. In areas where the surface is irradiated, thehydrophobic protecting groups are removed to define reaction regions.When an aqueous or other polar reactant solution is deposited in thereaction region, it will have a relatively large wetting angle with thesubstrate surface so that by adjusting the amount deposited, one canensure no flow to adjacent regions.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OP THE DRAWINGS

FIG. 1 is a generalized diagram illustrating the invention;

FIG. 2 is a flow chart illustrating the treatment steps performed insynthesizing an array of various polymers;

FIG. 3 is a mapping of a resulting array of polymers;

FIG. 4 a to 4 c illustrate the arrangement of three channel blocktemplates in six process steps employed to synthesize 64 millionhexapeptides from a 20 amino acid basis set;

FIG. 5 a is a top view and FIG. 5 b is a cross-sectional view of a firstembodiment of a device used to synthesize arrays of polymer sequences;

FIG. 6 is a cross-sectional view of an embodiment containing a pressurechamber for holding a substrate against a channel block;

FIGS. 7 a and 7 b are top views of two of two different “fanned array”channel blocks;

FIG. 8 is a cross-sectional view of a channel block and associated flowports according to one embodiment of the invention;

FIG. 9 is a detailed cross-sectional view of the flow ports in a channelblock;

FIG. 10 is a diagram of a flow system used to deliver coupling compoundsand reagents to a flow cell;

FIGS. 11 a and 11 b show an apparatus used to transfer a substrate fromone channel block to another;

FIG. 12 is a diagram of a multichannel solid-phase synthesizer;

FIGS. 13 a and 13 b illustrate alternative arrangements of the groovesin a channel block;

FIG. 14 is a schematic illustration of reaction pathways used to preparesome hydrophobic groups of the present invention;

FIGS. 15 a and 15 b illustrate a microvalve device;

FIGS. 16 a and 16 b Illustrate an alternative embodiment of theinvention;

FIG. 17 is a mapping of expected fluorescent intensities with asubstrate selectively exposed to fluorescent dye;

FIG. 18 is a mapping of actual fluorescence intensity versus location;

FIG. 19 is a mapping of fluorescence intensity versus location on aslide having four different peptides synthesized thereon;

FIG. 20 is a mapping of fluorescence intensity versus locationindicating fluorescein binding on 400 micron wide photolyzed regionsperpendicular to 100 micron flow paths;

FIG. 21 is a mapping of fluorescence intensity versus location for asubstrate containing octanucleotides, heptanucleotides, andhexanucleotides and incubated with an oligonucleotide complimentary tothe octanucleotide; and

FIG. 22 is a magnified version of FIG. 21.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Contents

I. Glossary

II. General

III. Methods for Mechanical Delivery of Reagents

IV. Flow Channel Embodiments

V. Spotting Embodiments

VI. Alternative Embodiments

VII. Examples

-   -   A. Leak Testing    -   B. Formation of YGGFL    -   C. 100 Micron Channel Block    -   D. Channel Matrix Hybridization Assay

VIII. Conclusion

I. Glossary

The following terms are intended to have the following general meaningsas they are used herein:

-   1. Ligand: A ligand in a molecule that is recognized by a receptor.    Examples of ligands that can be investigated by this invention    include, but are not restricted to, agonists and antagonists for    cell membrane receptors, toxins and venoms, viral epitopes,    hormones, opiates, steroids, peptides, enzyme substrates, cofactors,    drugs, lectins, sugars, oligonucleotides, nucleic acids,    oligosaccharides, and proteins.-   2. Monomer: A monomer is a member of the set of small molecules    which are or can be joined together to form a polymer or a compound    composed of two or more members. The set of monomers includes but is    not restricted to, for example, the set of common L-amino acids, the    set of D-amino acids, the set of synthetic and/or natural amino    acids, the set of nucleotides and the set of pentoses and hexoses.    The particular ordering of monomers within a polymer is referred to    herein as the “sequence” of the polymer. As used herein, monomers    refers to any member of a basis set for synthesis of a polymer. For    example, dimers of the 20 naturally occurring L-amino acids form a    basis set of 400 monomers for synthesis of polypeptides. Different    basis sets of monomers may be used at successive steps in the    synthesis of a polymer. Furthermore, each of the sets may include    protected members which are modified after synthesis. The invention    in described herein primarily with regard to the preparation of    molecules containing sequences of monomers such as amino acids, but    could readily be applied in the preparation of other polymers. Such    polymers include, for example, both linear and cyclic polymers of    nucleic acids, polysaccharides, phospholipids, and peptides having    either α-, β-, or ω-amino acids, heteropolymers in which a known    drug is covalently bound to any of the above, polynucleotides,    polyurethanes, polyesters, polycarbonates, polyureas, polyamides,    polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides,    polyacetates, or other polymers which will be apparent upon review    of this disclosure. Such polymers are “diverse” when polymers having    different monomer sequences are formed at different predefined    regions of a substrate. Methods of cyclization and polymer reversal    of polymers are disclosed in copending application Ser. No. 796,727,    filed Nov. 22, 1991, entitled “POLYMER REVERSAL ON SOLID SURFACES,”    incorporated herein by reference for all purposes.-   Peptide: A peptide is a polymer in which the monomers are alpha    amino acids and are joined together through amide bonds,    alternatively referred to an a polypeptide. Amino acids may be the    L-optical isomer or the D-optical isomer. Peptides are two or more    amino acid monomers long and are often more than 20 amino acid    monomers long. Standard abbreviations for amino acids are used    (e.g., P for proline). These abbreviations are included in Stryer,    Biochemistry, Third Ed., 1988, which in incorporated herein by    reference for all purposes.-   Receptor: A receptor is a molecule that has an affinity for a    ligand. Receptors may be naturally-occurring or manmade molecules.    They can be employed in their unaltered state or an aggregates with    other species. Receptors may be attached, covalently or    noncovalently, to a binding member, either directly or via a    specific binding substance. Examples of receptors which can be    employed by this invention include, but are not restricted to,    antibodies, cell membrane receptors, monoclonal antibodies and    antisera reactive with specific antigenic determinants, viruses,    cells, drugs, polynucleotides, nucleic acids, peptides, cofactors,    lectins, sugars, polysaccharides, cellular membranes, and    organelles. Receptors are sometimes referred to in the art as    anti-ligands. As the term receptors is used herein, no difference in    meaning is intended. A “Ligand Receptor Pair” is formed when two    molecules have combined through molecular recognition to form a    complex.

Specific examples of receptors which can be investigated by thisinvention include but are not restricted to:

-   -   a) Microorganism receptors: Determination of ligands that bind        to microorganism receptors such as specific transport proteins        or enzymes essential to survival of microorganisms would be a        useful tool for discovering new classes of antibiotics. Of        particular value would be antibiotics against opportunistic        fungi, protozoa, and bacteria resistant to antibiotics in        current use.    -   b) Enzymes: For instance, a receptor can comprise a binding site        of an enzyme such as an enzyme responsible for cleaving a        neurotransmitter; determination of ligands for this type of        receptor to modulate the action of an enzyme that cleaves a        neurotransmitter is useful in developing drugs that can be used        in the treatment of disorders of neurotransmission.    -   c) Antibodies: For instance, the invention may be useful in        investigating a receptor that comprises a ligand-binding site on        an antibody molecule which combines with an epitope of an        antigen of interest; determining a sequence that mimics an        antigenic epitope may lead to the development of vaccines in        which the immunogen in based on one or more of such sequences or        lead to the development of related diagnostic agents or        compounds useful in therapeutic treatments such as for        autoimmune diseases (e.g., by blocking the binding of the “self”        antibodies).    -   d) Nucleic Acids: Sequences of nucleic acids may be synthesized        to establish DNA or RNA binding sequences that act as receptors        for synthesized sequence.    -   e) Catalytic Polypeptides: Polymers, preferably antibodies,        which are capable of promoting a chemical reaction involving the        conversion of one or more reactants to one or more products.        Such polypeptides generally include a binding site specific for        at least one reactant or reaction intermediate and an active        functionality proximate to the binding site, which functionality        is capable of chemically modifying the bound reactant. Catalytic        polypeptides and others are described in, for example, PCT        Publication No. W 90/05746, WO 90/05749, and WO 90/05785, which        are incorporated herein by reference for all purposes.    -   f) Hormone receptors: Determination of the ligands which bind        with high affinity to a receptor such as the receptors for        insulin and growth hormone is useful in the development of, for        example, an oral replacement of the daily injections which        diabetics must take to relieve the symptoms of diabetes or a        replacement for growth hormone. Other examples of hormone        receptors include the vasoconstrictive hormone receptors;        determination of ligands for these receptors may lead to the        development of drugs to control blood pressure.    -   g) Opiate receptors: Determination of ligands which bind to the        opiate receptors in the brain in useful in the development of        less-addictive replacements for morphine and related drugs.

-   5. Substrate: A material having a rigid or semi-rigid surface. In    many embodiments, at least one surface of the substrate will be    substantially flat, although in some embodiments it may be desirable    to physically separate synthesis regions for different polymers    with, for example, wells, raised regions, etched trenches, or the    like. In some embodiments, the substrate itself contains wells,    trenches, flow through regions, etc. which form all or part of the    synthesis regions. According to other embodiments, small beads may    be provided on the surface, and compounds synthesized thereon may be    released upon completion of the synthesis.

-   6. Channel Blocks: A material having a plurality of grooves or    recessed regions on a surface thereof. The grooves or recessed    regions may take on a variety of geometric configurations, including    but not limited to stripes, circles, serpentine paths, or the like.    Channel blocks may be prepared in a variety of manners, including    etching silicon blocks, molding or pressing polymers, etc.

-   7. Protecting Group: A material which is bound to a monomer unit and    which may be selectively removed therefrom to expose an active site    such as, in the specific example of an amino acid, an amine group.    Specific examples of photolabile protecting groups are discussed in    Fodor et al., PCT Publication No. WO 92/10092 (previously    incorporated by references and U.S. Ser. No. 07/971,181 filed Nov.    2, 1992 incorporated herein by reference for all purposes.

-   8. Predefined Region: A predefined region is a localized area on a    substrate which is, was, or is intended to be used for formation of    a selected polymer and is otherwise referred to herein in the    alternative as “reaction” region, a “Selected” region, or simply a    “region.” The predefined region may have any convenient shape, e.g.,    circular, rectangular, elliptical, wedge-shaped, etc. in some    embodiments, a predefined region and, therefore, the area upon which    each distinct polymer sequence in synthesized is smaller than about    1 cm², more preferably less than 1 mm², and still more preferably    less than 0.5 mm². In most preferred embodiments the regions have an    area less than about 10,000 μm² or, more preferably, less than 100    μm². Within these regions, the polymer synthesized therein is    preferably synthesized in a substantially pure form.

-   9. Substantially Pure: A polymer is considered to be “substantially    pure” within a predefined region of a substrate when it exhibits    characteristics that distinguish it from other predefined regions.    Typically, purity will be measured in terms of biological activity    or function an a result of uniform sequence. Such characteristics    will typically be measured by way of binding with a selected ligand    or receptor. Preferably the region is sufficiently pure such that    the predominant species in the predefined region is the desired    sequence. According to preferred aspects of the invention, the    polymer is at least 5% pure, more preferably more than 10% to 20%    pure, more preferably more than 80% to 90% pure, and most preferably    more than 95% pure, where purity for this purpose refers to the    ratio of the number of ligand molecules formed in a predefined    region having a desired sequence to the total number of molecules    formed in the predefined region.    II. General

The invention can be used in variety of applications. For example, theinvention can be used as a synthesis tool (as for example in peptidesyntheses), as a screening tool (as for example in screening compoundlibraries for drug activity), or as a monitoring/diagnostic tool (as forexample in medical or environmental testing). In one specificembodiment, the invention is used for nucleic acid-based diagnostics.

As a synthesis tool, the present invention provides for the formation ofarrays of large numbers of different polymer sequences. According to apreferred embodiment, the invention provides for the synthesis of anarray of different peptides or oligonucleotides in selected regions of asubstrate. Such substrates having the diverse sequences formed thereonmay be used in, for example, screening studies to evaluate theirinteraction with receptors such as antibodies and nucleic acids. Forexample, in preferred embodiments the invention provides for screeningof peptide to determine which if any of a diverse set of peptides has astrong binding affinity with a receptor and, in most preferredembodiments, to determine the relative binding affinity of variouspeptides with a receptor of interest.

Such diverse polymer sequences are preferably synthesized on a singlesubstrate. By synthesizing the diverse polymer sequences on a singlesubstrate, processing of the sequences to evaluate characteristics suchas relative binding affinity is more easily conducted. By way ofexample, when an array of peptide sequences (or a library of othercompounds) is to be evaluated to determine the peptides' relativebinding affinity to a receptor, the entire substrate and, therefore, allor a group of the polymer sequences may be exposed to an appropriatelylabelled receptor and evaluated simultaneously.

In some embodiments, the present invention can be employed to localizeand, in some cases, immobilize vast collections of synthetic chemicalcompounds or natural product extracts. In such methods, compounds aredeposited on predefined regions of a substrate. The reaction of theimmobilized compound (or compounds) with various test compositions suchas the members of the chemical library or a biological extract aretested by dispensing small aliquots of each member of the library orextract to a different region. Competitive assays or other well-knowntechniques can be used to identify a desired activity. As an example, alarge collection of human receptors in deposited on a substrate, one ineach region to form an array. A plant/animal extract is then screenedfor binding to various receptors of the array.

The present invention has certain features in common with the “lightdirected” methods described in U.S. Pat. No. 5,143,854, previouslyincorporated by reference. The light directed methods discussed in the'854 patent involve activating predefined regions of the substrate andthen contacting the substrate with a preselected monomer solution. Thepredefined regions can be activated with a light source shown through amask (much in the manner of photolithography techniques used inintegrated circuit fabrication). Other regions of the substrate remaininactive because they are blocked by the mask from illumination. Thus, alight pattern defines which regions of the substrate react with a givenmonomer. By repeatedly activating different sets of predefined regionsand contacting different monomer solutions with the substrate, a diversearray of polymers is produced on the substrate. Of course, other stepssuch as washing unreacted monomer solution from the substrate can beused as necessary.

In the present invention, a mechanical device or physical structuredefines the regions which are available to react with a given monomer.In some embodiments, a wall or other physical barrier is used to block agiven monomer solution from contacting any but a few selected regions ofa substrate. In other embodiments, the amount of the monomer (or other)solution deposited and the composition of the substrate act to separatedifferent monomer solutions on the substrate. This permits differentmonomers to be delivered and coupled to different regions simultaneously(or nearly simultaneously) and reduces the number of separate washingand other reaction steps necessary to form an array of polymers.Further, the reaction conditions at different activated regions can becontrolled independently. Thus, the reactant concentrations and otherparameters can be varied independently from reaction site to reactionsite, to optimize the procedure.

In alternative preferred embodiments of the present invention, light oranother activator is used in conjunction with the physical structures todefine reaction regions. For example, a light source activates variousregions of the substrate at one time and then a mechanical systemdirects monomer solutions to different activated regions, in parallel.

III. Methods for Mechanical Delivery of Reagents

In preferred embodiments of the present invention, reagents aredelivered to the substrate by either (1) flowing within a channeldefined on predefined regions or (2) “spotting” on predefined regions.However, other approaches, as well as combinations of spotting andflowing, may be employed. In each instance, certain activated regions ofthe substrate are mechanically separated from other regions when themonomer solutions are delivered to the various reaction sites.

A typical “flow channel” method of the present invention can generallybe described as follows. Diverse polymer sequences are synthesized atselected regions of a substrate by forming flow channels on a surface ofthe substrate through which appropriate reagents flow or in whichappropriate reagents are placed. For example, assume a monomer “A” is tobe bound to the substrate in a first group of selected regions. Ifnecessary, all or part of the surface of the substrate in all or a partof the selected regions is activated for binding by, for example,flowing appropriate reagents through all or some of the channels, or bywashing the entire substrate with appropriate reagents. After placementof a channel block on the surface of the substrate, a reagent having themonomer A flows through or is placed in all or some of the channel(s).The channels provide fluid contact to the first selected regions,thereby binding the monomer A on the substrate directly or indirectly(via a linker) in the first selected regions.

Thereafter, a monomer B is coupled to second selected regions, some ofwhich may be included among the first selected regions. The secondselected regions will be in fluid contact with a second flow channel(s)through translation, rotation, or replacement of the channel block onthe surface of the substrate; through opening or closing a selectedvalve; or through deposition of a layer of photoresist. If necessary, astep is performed for activating at least the second regions.Thereafter, the monomer B is flowed through or placed in the second flowchannel(s), binding monomer B at the second selected locations. In thisparticular example, the resulting sequences bound to the substrate atthis stage of processing will be, for example, A, B. and AB. The processis repeated to form a vast array of sequences of desired length at knownlocations on the substrate.

After the substrate is activated, monomer A can be flowed through someof the channels, monomer B can be flowed through other channels, amonomer C can be flowed through still other channels, etc. In thismanner, many or all of the reaction regions are reacted with a monomerbefore the channel block must be moved or the substrate must be washedand/or reactivated. By making use of many or all of the availablereaction regions simultaneously, the number of washing and activationsteps can be minimized.

Various embodiments of the invention will provide for alternativemethods of forming channels or otherwise protecting a portion of thesurface of the substrate. For example, according to some embodiments, aprotective coating such as a hydrophilic or hydrophobic coating(depending upon the nature of the solvent) is utilized over portions ofthe substrate to be protected, sometimes in combination with materialsthat facilitate wetting by the reactant solution in other regions. Inthis manner, the flowing solutions are further prevented from passingoutside of their designated flow paths.

The “spotting” embodiments of the present invention can be implementedin much the same manner as the flow channel embodiments. For example, amonomer A can be delivered to and coupled with a first group of reactionregions which have been appropriately activated. Thereafter, a monomer Bcan be delivered to and reacted with a second group of activatedreaction regions. Unlike the flow channel embodiments described above,reactants are delivered by directly depositing (rather than flowing)relatively small quantities of them in selected regions. In some steps,of course, the entire substrate surface can be sprayed or otherwisecoated with a solution. In preferred embodiments, a dispenser moves fromregion to region, depositing only as much monomer as necessary at eachstop. Typical dispensers include a micropipette to deliver the monomersolution to the substrate and a robotic system to control the positionof the micropipette with respect to the substrate. In other embodiments,the dispenser includes a series of tubes, a manifold, an array ofpipettes, or the like so that various reagents can be delivered to thereaction regions simultaneously.

IV. Flow Channel Embodiments

FIG. 1 illustrates an example of the invention. In this particularexample, monomers and dimers of the monomer group A, B, C, and D are tobe bound at selected regions of the substrate. The substrate may bebiological, nonbiological, organic, inorganic, or a combination of anyof these, existing as particles, strands, precipitates, gels, sheets,tubing, spheres, containers, capillaries, pads, slices, films, plates,slides, etc. The substrate may have any convenient shape, such as adisc, square, sphere, circle, etc. The substrate is preferably flat butmay take on a variety of alternative surface configurations. Forexample, the substrate may contain raised or depressed regions on whichthe synthesis takes place.

The substrate and its surface form a support on which to carry out thereactions described herein. These monomers are bound using first flowchannel paths x₁, x₂, x₃, and x₄, which are formed or placed on oradjacent the substrate in a first orientation, and second flow channelpaths y₁, y₂, y₃, and y₄, which are formed or placed on or adjacent thesubstrate in a second orientation. The second flow channel pathsintersect at least a part of the first flow channel paths. The flowchannels are formed according to techniques which are described ingreater detail elsewhere herein.

Initially the substrate is subjected to one or more preliminarytreatments such as, for example, cleaning and the optional placement of“linker” molecules on the surface thereof. The substrate may also beprovided with various active groups, common monomer sequences which willform a part of the polymers, or the like.

Thereafter, in a first coupling step, one or more of the flow channelsare provided with the first monmer A, which binds through covalent bondsor otherwise to the substrate (directly or indirectly) where the flowchannel contacts the substrate. In the particular example shown in FIG.1, the flow channels x₁ and x₂ are utilized, binding the monomer A tothe substrate along the entire length of the substrate adjacent to thex₁ and x₂ channels. Each coupling step may in some embodiments becomposed of a variety of substeps. For example, each coupling step mayinclude one or more substeps for washing, chemical activation, or thelike.

Thereafter or concurrently therewith, an shown in FIG. 2, a secondmonomer a is provided to selected flow channels and the monomer B bindsto the substrate where the second flow channels provide contacttherewith. In the particular example shown in FIG. 2, monomer B is boundalong channels x₃ and x₄. When the monomers A and B flow through theirrespective flow channels simultaneously, only a single process step isrequired to perform two coupling steps simultaneously. As used herein, a“process step” refers to the injection of one or more channels with oneor more reagents. A “coupling step” refers to the addition of a monomerin a polymer.

Processing thereafter continues in a similar manner with monomers C andD in the manner shown in the flow diagram of FIG. 2, with monomer Cbeing bound in the flow channels y₁ and y₂, and D being bound in theflow channels y₃ and y₄. Preferably, monomers C and D are directedthrough the flow channels y₁ to y₄ simultaneously whereby two couplingsteps are performed with a single process step. Light regions in FIG. 1indicate the intersections of the resulting flow paths.

FIG. 3 illustrates the mapping of sequences formed using the aboveillustrated steps. As shown therein, the sequences A, B, C, D, AD, BD,AC, and BC have been formed using only two process steps. Accordingly,it in seen that the process provides for the synthesis of vast arrays ofpolymer sequences using only a relatively few process steps. By way offurther example, it is necessary to use only two process steps to formall of the 4²=16 dimers of a four-monomer basis set. By way of furtherexample, to form all 4³ octomers of a four-monomer basis set, it isnecessary to provide only 256 flow channels oriented in the “x”direction, and 256 flow channels oriented in the “y” direction, with atotal of eight coupling steps.

The power of the technique in further illustrated by synthesizing thecomplete array of six hexamer peptides from a 20 amino acid basis set.This array will include 20⁶ or 64,000,000 regions defining 64,000,000different peptides and can be formed in only six process steps. Further,the method requires only three different templates, one having 20parallel channels, a second having 400 channels each {fraction (1/20)}thas wide as the first, and a third having 8000 channels each {fraction(1/20)}th as wide as the second. Each template will be used in twoprocess steps, each at an orientation at 90 degrees with respect to theother as illustrated in FIG. 4. With the first template, the substrateis activated and then solutions of each of the 20 amino acid basis set(or other 20 member basis set) are flowed over and reacted on adifferent predefined stripe in a first orientation. This is the firstprocess step and includes 20 coupling or attachment steps, which can beperformed simultaneously. Next, the entire substrate is again activatedand the first template is placed in a second orientation, perpendicularto the first (FIG. 4 a). The 20 amino acid solutions are then flowedalong 20 new predefined stripes (each perpendicular to the original setof stripes). In each of these two process steps, the 20 predefinedregions (the stripes along the flow channels) are first activated andthen contacted with the individual monomers so that all 20 stripes arereacted before the next activation step is necessary. In other words, 20coupling steps are conducted in parallel, greatly reducing the number ofnecessary activation steps.

The four remaining coupling steps employ the second and third templates.In the third and fourth process steps (FIG. 4 b), 20 channels aredevoted to each monomer, and in the fifth and sixth process steps (FIG.4 c), 400 channels are devoted to each monomer. As with the first twosteps, the entire substrate undergoes reaction during a single processstep. Thus, only six process steps (requiring a total of about 24 hours)are required to produce the entire library of 64,000,000 peptidehexamers. In a different embodiment, a single template having 8000channels to control delivery (e.g. 400 channels for each of the 20 aminoacids in the first round) can produce the full library of hexamers withonly a single rotation step. Thus, the present invention offersextremely rapid methods of preparing diverse polymer arrays.

FIGS. 5 a and 5 b illustrate details of a first embodiment of a deviceused for performing the synthesis steps described above. In particular,FIG. 5 a illustrates the device in top view, while FIG. 5 b illustratesthe device in cross-sectional side view. In the particular embodimentshown in FIG. 5, the device is used to synthesize polymer sequences onsubstrate 401. Substrate 401 is coupled to a rotating stage 403 andremovably held by clamp 405 to channel block 407. Channel block 407 hasetched therein a plurality of channels 409 in the form of stripestherein. Each channel is provided with a flow inlet 411 and an outlet413. A vacuum source 415 is applied to one or more of the outlets 413,while a pipettor 417 is slidably mounted on arm 419 to deliver selectedreagents from reservoir(s) 421 to selected flow inlets 411.

The details of a second preferred embodiment are shown in FIGS. 6-11.FIG. 6 displays an apparatus for holding a substrate 111 in placeagainst a channel block 209 by evenly distributing pressure over thesubstrate in a pressure chamber 101. Pressurized gas in admitted throughgas pressure inlet 103 to provide clamping pressure to immobilize thesubstrate while fluids are flowed from fluid flow inlet 115, throughchannel 123, and out fluid outlet 117. The upper and lower portions ofthe pressure chamber housing 105 and 125 are held together by nuts 121and bolts 104. Of course, other means such an clamps can be used to holdthe pressure chamber housing portions together.

FIG. 7 illustrates preferred flow path configurations in channel blocksof the present invention. As shown in FIGS. 7 a, fluid delivery sites127, 129, 131, 133, 135, and 137 are connected to channels leading toreaction region 141. A similar arrangement in shown for comparision inFIG. 7 b where the orientation of the flow channels in the reactionregions is shifted by 90 degrees on a rectangular channel block. Vacuumports 145 and 146 to an external vacuum line are provided so thatsubstrate position is maintained during fluid flow.

The channels shown in FIGS. 7 a and 7 b form a “fanned channel array” onchannel block 139 in a manner analogous to that of the lead patternemployed in integrated circuits. This provides significantly increasedseparation of fluid delivery points in comparison to the high density ofchannels in the reaction region. In a 2 inch by 3 inch substrate, atleast about a 4:1 increase in spatial separation typically can beattained by the fanned arrangement. Thus, if the channels in thereaction regions are separated by 200 microns, the delivery ports can beseparated by 0.8 mm.

The spatial separation can be further increased by staggering thedelivery ports as shown for ports 127, 129, and 131. This can provide anadditional channel separation of at least about 3:1. Thus, for thechannels separated by 200 microns, a staggered fanned array provides 2.4m separation between the delivery ports. Thus, fluid can be delivered toa high-density array of of channels in the reaction region from standard1.6 mm Teflon™ tubing. If additional spacing is necessary, the substratesize can be increased, while preserving the reaction region size.

An shown in FIG. 8, the fluid delivery ports are accessed from holes inthe back surface of a stabilizing plate 108 on the channel block. Thestabilizing plate, which is preferably made from fused pyrex, providesstructural integrity to the channel block during clamping in thepressure chamber. It may also provide a means to access the channelblock ports and reduce leakage between ports or channels. In preferredembodiments, the channels 123 of the channel block are formed on a wafer106 which generally may be any machinable or cast material, andpreferably may be etched silicon or a micromachined ceramic. In otherembodiments, the channel block is pressure-formed or injection-moldedfrom a suitable polymer material. The entire channel block arrangementis mounted on a rigid channel block sub-plate 110 including a vacuumline 112, ports for fluid delivery lines 115, ports for fluid outletlines 117, and recessed regions for plug ends 151 and 153. With thisarrangement, the substrate can be clamped against the top surface of thechannel block (by vacuum or pressurized gas as shown in the embodimentof FIG. 6) while fluid enters and exits from below. Preferably, thesubplate will be made from a rigid material such as stainless steel oranodized aluminum.

Individual micro tubing connections can be made for each channel asshown in FIG. 9. Plug ends 151 are provided with a conical upper surfacethat mates with a conical recess 118 in pyrex stabilizing plate 108.Plug ends 151 also have a cylindrical lower surface that mates withcylindrical recess 116 in sub-plate 110. The subplate and stabilizingplate are held together by bolt 114 and threaded insert 112 or othersuitable engagement means.

FIG. 10 shows a fluid flow diagram of a preferred system of the presentinvention. The pressure is controlled at point 25 (P1) and point 21 (P2)so that a pressure drop (P1-P2) is maintained across the system.Coupling compounds such as activated monomers are supplied fromreservoirs 31, 32, and 33. Additional reagents are supplied fromreservoirs 15, 17, and 19. Of course, the monomer and coupling reagentreservoirs shown in FIG. 10 are representative of a potentially muchlarger series of reservoirs. The reagents and coupling compounds arecombined at nodes 27, 28, and 29 before being directed to channel block139. Mixing of the appropriate reagents and coupling compounds iscontrolled by valves at the nodes which are in turn controlled byelectronic control 23. Waste fluids that have been directed across thesubstrate are removed through line 35.

The system displayed in FIG. 10 allows control of all channels inparallel by regulating only a few variables. For example, a constantpressure gradient is maintained across all channels simultaneously byfixing P1 and P2. Thus, the flow rate in each channel is dependent uponthe cross-sectional area of the flow channel and the rheologicalproperties of the fluids. Because the channels have a uniformcross-section and because the coupling compounds are typically providedas dilute solutions of a single solvent, a uniform flow rate is createdacross all channels. With this system the coupling time in all channelscan be varied simultaneously by simply adjusting the pressure gradientacross the system. The valves of the system are preferably controlled bya single electronic output from control 23.

The fanned channel array design shown in FIG. 7 provides for twoseparate channel blocks to be used in successive process steps during achemical synthesis. One block forms a horizontal array on the solidsubstrate, while the other block forms a vertical array. To create amatrix of intersecting rows and columns of chemical compounds, the solidsubstrate is transferred from one block to the other during successiveprocess steps. While many experiments require only a single transferfrom one block to the other during a series of process steps, the fannedchannel array transfer block 75 illustrated in FIGS. 11 a and 11 bprovides one device for maintaining accurate registration of the solidsubstrate 71 relative to the channel blocks 79 during repeatedtransfers. In some embodiments, a single channel block can be used forhorizontal and vertical arrays by simply rotating it by 90 degrees asnecessary.

The transfer block is positioned with respect to the channel block sothat the dimensional characteristics of the solid substrate are not usedin the alignment. The transfer block 75 is aligned to the channel blockby kinematic mount 81 while vacuum is switched from vacuum line 83 onthe channel block to vacuum line 77 on the transfer block (during normaloperation, a vacuum holds the substrate against the channel block). Thesubstrate and transfer block are then moved and repositioned relative tothe second channel bock. Vacuum is then switched to the second channelblock, retaining the substrate in proper alignment. This way, accurateregistration can be assured between process steps regardless ofvariation in the dimensions of individual substrates. The transfer blocksystem also maintains alignment of the matrix area during transfers toand from the flow cell during experiments utilizing both mechanical andlight-directed process steps.

In some embodiments the channel block need not be utilized. Instead, insome embodiments, small “strips” of reagent are applied to the substrateby, for example, striping the substrate or channels therein with apipettor. Such embodiments bear some resemblance to the spottingembodiments of this invention. According to other embodiments thechannels will be formed by depositing a photoresist such as those usedextensively in the semiconductor industry. Such materials includepolymethyl methacrylate (PMMA) and its derivatives, and electron beamresists such as poly(olefin sulfones) and the like (more fully describedin Ghandi, “VLSI Fabrication Principles,” Wiley (1983) Chapter 10,incorporated herein by reference in its entirety for all purposes).According to these embodiments, a resist is deposited, selectivelyexposed, and etched, leaving a portion of the substrate exposed forcoupling. Theme steps of depositing resist, selectively removing resistand monomer coupling are repeated to form polymers of desired sequenceat desired locations.

In some embodiments, a resist can be used to activate certain regions ofthe substrate. Certain resist materials such as acid-generatingpolymers, for example, will release protons upon irradiation. Accordingto these embodiments, a substrate covered with such material isirradiated through a mask or otherwise selectively irradiated so thatthe irradiated regions of the substrate are exposed to acidicconditions. Acid-labile protecting group on the substrate or oligomerson the substrate are removed, leaving an activated region. At thispoint, all or part of the resist may be removed. In preferredembodiments, the resist will be removed only in the activated regions,so that the channels are formed at the activated regions. Alternatively,the resist can be removed from the entire substrate. In this case, aseparate channel block can then be contacted with the substrate todefine flow channels, or a conventional VLSIPS™ procedure can beemployed.

In preferred embodiments, the substrate is conventional glass, pyrex,quartz, any one of a variety of polymeric materials, or the like. Ofcourse, the substrate may be made from any one of a variety of materialssuch as silicon, polystyrene, polycarbonate, or the like. In preferredembodiments the channel block is made of silicon orpolychlorotrifluorethylene, such an material known under the trade nameKelF™ 80 made by 3M, although a wide variety of materials such aspolystyrene, polycarbonate, glass, elastomers such as Kalrez made byDuPont, various ceramics, stainless steel, or the like may be utilized.

The channels in the channel block are preferably made by machining,compression molding, injection molding, lithography, laser cutting, orthe like depending upon the material of interest. In some embodimentsemploying larger channel blocks, the raised portions of the channels inthe channel block are treated by lapping with lapping film (0.3 μmgrit). Such smooth surfaces provide good seals to the substrate withoutthe use of a sealant and, therefore, without the possibility of leavingsealant material on the substrate when rotating the channel block.Preferably, all operations are conducted at substantially ambienttemperatures and pressures.

A particularly preferred channel block is prepared by chemical etchingof polished silicon wafers. Chemical etching is a widely used techniquein integrated circuit fabrications. It can easily provide 60 or more 100micron channels on a 12.8 mm region of a polished silicon wafer. Evenafter etching, the top (unetched) surface regions of the wafer retainsthe very flat profile of the unetched wafer. Thus, close contact withthe substrate is ensured during flow cell operation.

In operation, the surface of the substrate is appropriately treated bycleaning with, for example, organic solvents, methylene chloride, DMF,ethyl alcohol, or the like. Optionally, the substrate may be providedwith appropriate linker molecules on the surface thereof. The linkermolecules may be, for example, aryl acetylene, ethylene glycol oligomerscontaining from 2-10 monomers or more, diamines, diacids, amino acids,or combinations thereof. Thereafter, the surface is provided withprotected surface active groups such as TBOC or FMOC protected aminoacids. Such techniques are well known to those of skill in the art.

Thereafter, the channel block and the substrate are brought into contactforming fluid-tight channels bounded by the grooves in the channel blockand the substrate. When the channel block and the substrate are incontact, a protecting group removal agent is, thereafter, directedthrough a first selected channel or group of channels by placing thepipettor on the flow inlet of the selected channel and, optionally, thevacuum source on the outlet of the channel. In the case of, for example,TBOC protected amino acids, this protecting group removal agent may be,for example, trifluoroacetic acid (TFA). This step is optionallyfollowed by steps of washing to remove excess TFA with, for example,dichloromethane (DCM).

Thereafter, a first amino acid or other monomer A is directed throughthe first selected flow channel. Preferably this first amino acid isalso provided with an appropriate protecting group such as TBOC, FMOC,NVOC, or the like. This step is also followed by appropriate washingsteps. The of deprotection/coupling steps employed in the first group ofchannels are concurrently with or thereafter repeated in additionalgroups of channels. In preferred embodiments, monomer A will be directedthrough the first group of channels, monomer B will be directed througha second group of flow channels, etc., so that a variety of differentmonomers are coupled on parallel channels of the substrate.

Thereafter, the substrate and the channel block are separated and,optionally, the entire substrate is washed with an appropriate materialto remove any unwanted materials from the points where the channelscontact the substrate.

The substrate and/or block is then, optionally, washed and translatedand/or rotated with the stage. In preferred embodiments, the substrateis rotated 90 degrees from its original position, although someembodiments may provide for greater or less rotation, such as from 0 to180 degrees. In other embodiments, such as those discussed in connectionwith the device shown in FIG. 7, two or more different channel blocksare employed to produce different flow patterns across the substrate.When the channel block is rotated, it may simultaneously be translatedwith respect to the substrate. “Translated” means any relative motion ofthe substrate and/or channel block, while “rotation” is intended torefer to rotation of the substrate and/or channel block about an axisperpendicular to the substrate and/or channel block. According to someembodiments the relative rotation is at different angles for differentstages of the synthesis.

The steps of deprotection, and coupling of amino acids or other monomersis then repeated, resulting in the formation of an array of polymers anthe surface of the substrate. For example, a monomer B may be directedthrough selected flow channels, providing the polymer AB atintersections of the channels formed by the channel block is the firstposition with the channels formed by the channel block after 90-degreerotation.

While rotation of the channel block is provided according to preferredembodiments of the invention, such rotation is not required. Forexample, by simply flowing different reagents through the channels,polymers having different monomer sequences may be formed. Merely by wayof a specific example, a portion of the channels may be filled withmonomer “A,” and a portion filled with monomer “B” in a first couplingstep. All or a portion of the first channels are then filled with amonomer “C,” and all or a portion of the second channels are filled witha monomer “D,” forming the sequences AB and CD. Such steps could be usedto form 100 sequences using a basis set of 10 monomers with a 100-groovechannel block.

It in another embodiment, the invention provides a multichannelsolid-phase synthesizer as shown in FIG. 12. In this embodiment, acollection of delivery lines such an a manifold or collection of tubes1000 delivers activated reagents to a synthesis support matrix 1002. Thecollection of tubes 1000 may take the form of a rigid synthesis blockmanifold which can be precisely aligned with the synthesis supportmatrix 1002. The support matrix contains a plurality of reaction regions1004 in which compounds may be immobilized or synthesized. In preferredembodiments, the reaction regions include synthesis frits, pads, resins,or the like.

The solutions delivered to the individual reactant regions of thesupport matrix flow through the reaction regions to waste disposalregions, recycling tank(s), separators, etc. In some embodiments, thereaction solutions simply pass through the reaction regions under theinfluence of gravity, while in other embodiments, the solutions arepulled or pushed through the reaction regions by vacuum or pressure.

The individual reaction regions 1004 of the support matrix are separatedfrom one another by walls or gaskets 1006. These prevent the reactantsolution in one reaction region from moving to and contaminatingadjacent reaction regions. In one embodiment, the reaction regions aredefined by tubes which may be filled with resin or reaction mixture. Thegasketing allows close contact between the support matrix 1002 and a“mask” (not shown). The mask serves to control delivery of a first groupreactant solutions through predetermined lines (tubes) to a first set ofreaction regions. By ensuring close contact between the delivery tubes1000, the mask, and the support matrix 1002, the probability thatreaction solutions will be accidently added to the wrong reaction siteis reduced.

After each process step, the mask can be changed so that a new groupreactants is delivered to a new set of reaction regions. In this manner,a combinatorial strategy can be employed to prepare a large array ofpolymers or other compounds. In other embodiments, mechanisms other thanmasks can be employed to block the individual delivery tubes. Forexample, an array of control valves within the tubes may be suitable forsome embodiments.

By adjusting the thickness of the synthesis support matrix, the quantityof immobilized material in the reaction regions can be controlled. Forexample, relatively thin support synthesis matrices can be used toproduce small amounts of surface bound oligomers for analysis, whilethicker support matrices can be used to synthesize relatively largequantities of oligomers which can be cleaved from the support forfurther use. In the latter embodiment, a collector having dimensionsmatching the individual synthesis supports can be employed to collectoligomers that are ultimately freed from the reaction matrix.

To illustrate the ability of this system to synthesize numerouspolymers, a square synthesis matrix measuring 10 cm along each side andhaving 5 mm reaction regions separated by 5 mm wide gaskets provides 100individual syntheses sites (reaction regions). By reducing the size ofthe reaction regions to 2.5 mm on each side, 400 reactions regionsbecome available.

While linear grooves are shown herein in the preferred aspects of theinvention, other embodiments of the invention will provide for circularrings or other shapes such as circular rings with radial grooves runningbetween selected rings. According to some embodiments, channel blockswith different geometric configurations will be used from one step tothe next, such as circular rings in one step and linear stripes in thenext. FIG. 13 a illustrates one of the possible arrangements in whichthe channels 409 are arranged in a serpentine arrangement in the channelblock 407. Through appropriate translation and/or rotation of thechannel block, polymers of desired monomer sequence are formed at theintersection of the channels during successive polymer additions, suchas at location 501, where the intersection of a previous or subsequentset of channels is shown in dashed lines. FIG. 13 b illustrates anotherarrangement in which channels (in this case without flow paths 413) areprovided in a linear arrangement, with groups 503 and 505 located inadjacent regions of the substrate and extending only a portion of thesubstrate length.

In some embodiments of the invention, the various reagents, such asthose containing the various monomers, are not pumped through theapertures 413. Instead, the reagent is placed in one of the grooves,ouch as the grooves 409 shown in FIG. 13 b, filling the groove. Thesubstrate is then placed on top of the channel block, and the exposedportions of the substrate are permitted to react with the materials inthe grooves. In preferred embodiments, the channels are of the samewidth as the raised regions between the channels. According to theseembodiments, the substrate may then be moved laterally by one channelwidth or an integer multiple of a channel width, permitting reactionwith and placement of monomers on the regions between the channels in aprevious coupling step. Thereafter, the substrate or channel block willbe rotated for the next series of coupling steps.

In preferred embodiments, the process is repeated to provide more than10 different polymer sequences on the surface of the substrate. In morepreferred embodiments, the process is repeated to provide more than 10²,10³, 10⁴, 10⁵, 10⁶, or more polymer sequences on a single substrate. Insome embodiments the process is repeated to provide polymers with as fewas two monomers, although the process may be readily adapted to formpolymers having 3, 4, 5, 6, 10, 15, 20, 30, 40, 50, 75, 100 or moremonomers therein.

According to preferred embodiments, the array of polymer sequences isutilized in one or more of a variety of screening processes, one ofwhich is described in copending application U.S. Ser. No. 796,947, filedon Nov. 22, 1991 and incorporated herein by reference for all purposes.For example, according to one embodiment, the substrate is then exposedto a receptor of interest such as an enzyme or antibody. According topreferred embodiments, the receptor is labelled with fluorescein, orotherwise labelled, so as to provide for easy detection of the locationat which the receptor binds. According to some embodiments, the channelblock is used to direct solutions containing a receptor over asynthesized array of polymers. For example, according to someembodiments the channel block is used to direct receptor solutionshaving different receptor concentrations over regions of the substrate.

According to most preferred embodiments, amplification of the signalprovided by way of fluorescein labelling is provided by exposing thesubstrate to the antibody of interest, and then exposing the substrateto a labelled material which is complementary to the antibody ofinterest and which preferably binds at multiple locations of theantibody of interest. For example, in one specific embodiment, if amouse antibody is to be studied, a labelled second antibody may beexposed to the substrate which is, for example, goat antimouse. Suchtechniques are described in PCS Publication No. WO92/10092, previouslyincorporated herein by reference.

V. Spotting Embodiments

According to some embodiments, monomers (or other reactants) aredeposited from a dispenser in droplets that fill predefined regions. Forexample, in a single coupling step, the dispenser deposits a firstmonomer in a series of predefined regions by moving over a first region,dispensing a droplet, moving to a second region, dispensing a droplet,and so on until the each of the selected regions has received themonomer. Next the dispenser deposits a second monomer in a second seriesof predefined regions in much the same manner. In some embodiments, morethan one dispenser may be used so that more than one monomer aresimultaneously deposited. The monomers may react immediately on contactwith the reaction regions or may require a further activation step, suchas the addition of catalyst. After some number of monomers have beendeposited and reacted in predefined regions throughout the substrate,the unreacted monomer solution is removed from the substrate. Thiscompletes a first process step.

For purposes of this embodiment, the spacing between the individualreaction regions of the substrate preferably will be less than about 3mm, and more preferably between about 5 and 100 μm. Further, the angularrelation between the regions is preferably consistent to within 1 degreeand more preferably to within 0.1 degree. Preferably, the substrate willinclude at least about 100 reaction regions, more preferably at leastabout 1000 reaction regions, and most preferably at least about 10,000reaction regions. Of course, the density of reaction regions on thesubstrate will vary. In preferred embodiments, there are at least about1000 reaction regions per cm² of substrate, and more preferably at leastabout 10,000 regions per cm².

To deposit reactant droplets consistently at precisely specifiedregions, a frame of reference common to the delivery instrument and thesubstrate is required. In other words, the reference coordinates of theinstrument must be accurately mapped onto the reference coordinates ofthe substrate. Ideally, only two reference points on the substrate arenecessary to map the array of polymer regions completely. The dispenserinstrument locates these reference points and then adjusts its internalreference coordinates to provide the necessary mapping. After this, thedispenser can move a particular distance in a particular direction andbe positioned directly over a known region. of course, the dispenserinstrument must provide precisely repeatable movements. Further, theindividual regions of the array must not move with respect to thereference marks on the substrate after the reference marks have beenformed. Unfortunately, pressing or other mechanical operations commonlyencountered during fabrication and use of a substrate can warp thesubstrate such that the correspondence between the reference marks andthe reaction regions is altered.

Thus, in preferred embodiments, a substrate containing both “global” and“local” reference marks is employed. In preferred embodiments, twoglobal reference marks are conveniently located on the substrate todefine the initial frame of reference. When these points are located,the dispenser instrument has an approximate map of the substrate and thepredefined regions therein. To assist in locating the exact position ofthe regions, the substrate is further subdivided into local frames ofreference. Thus, in an initial, “course” adjustment, the dispenser inpositioned within one of the local frames of reference. Once in thelocal region, the dispensing instrument looks for local reference marksto define further a local frame of reference. From these, the dispensermoves exactly to the reaction region where the monomer is deposited. Inthis manner, the effects of warpage or other deformation can beminimized. The number of local reference marks is determined by theamount of deformation expected in the substrate. If the substrate issufficiently rigid so that little or no deformation will occur, very fewlocal reference marks are required. If substantial deformation isexpected, however, more local reference marks are required.

In order to locate the appropriate reference point initially and alignthe dispenser with respect to it, a vision or blind system may beemployed. In a vision system, a camera is rigidly mounted to thedispenser nozzle. When the camera locates the reference point(s), thedispenser is known to be a fixed distance and direction away from thepoint, and a frame of reference is established. Blind systems of thepresent invention locate the reference point(s) by capacitive,resistive, or optical techniques, for example. In one example of anoptical technique, a laser beam is transmitted through or reflected fromthe substrate. When the beam encounters a reference mark, a change inlight intensity is detected by a sensor. Capacitive and resistivetechniques are similarly applied. A sensor registers a change incapacitance or resistivity when a reference point is encountered.

Starting at a single reference point, the dispenser is translated fromone reaction region to other regions of the substrate by a correctdistance in the correct direction (this in the “dead reckoning”navigational technique). At each stop, the dispenser deposits correctlymetered amounts of monomer. Analogous systems widely used in themicroelectronic device fabrication and testing arts can move at rates ofup to 3-10 stops per second. The translational (X-Y) accuracy of suchsystems is well within 1 μm.

Translational mechanisms for moving the dispenser are preferablyequipped with closed loop position feedback mechanisms (encoders) andhave insignificant backlash and hysteresis. In preferred embodiments,the translation mechanism has a high resolution, i.e. better than onemotor tick per encoder count. Further, the electromechanical mechanismpreferably has a high repeatability relative to the reaction regiondiameter travel distance (typically ±1 μm or better).

To deposit a drop of monomer solution on the substrate accurately, thedispenser nozzle must be placed a correct distance above the surface. Inone embodiment, the dispenser tip preferably will be located about 5-50μm above the substrate surface when a five nanoliter drop is released.More preferably, the drop will be about 10 μm above the substratesurface when the drop is released. The degree of control necessary toachieve such accuracy in attained with a repeatable high-resolutiontranslation mechanism of the type described above. In one embodiment,the height above the substrate is determined by moving the dispensertoward the substrate in small increments, until the dispenser tiptouches the substrate. At this point, the dispenser is moved away fromthe surface a fixed number of increments which corresponds to a specificdistance. From there the drop is released to the cell below. Preferably,the increments in which the dispenser moves less than about 5 μm andmore preferably less than about 2 μm.

In an alternative embodiment, the dispenser nozzle is encircled by asheath that rigidly extends a fixed distance beyond the dispenser tip.Preferably, this distance corresponds to the distance the solution dropwill fall when delivered to the selected reaction region. Thus, when thesheath contacts the substrate surface, the movement of the dispenser ishalted and the drop is released. It is not necessary in this embodimentto move the dispenser back, away from the substrate, after contact ismade. The point of contact with the surface can be determined by avariety of techniques such as by monitoring the capacitance orresistance between the tip of the dispenser (or sheath) and thesubstrate below. A rapid change in either of these properties isobserved upon contact with the surface.

To this point, the spotting system has been described only in terms oftranslational movements. However, other systems may also be employed. Inone embodiment, the dispenser is aligned with respect to the region ofinterest by a system analogous to that employed in magnetic and opticalstorage media fields. For example, the region in which monomer is to bedeposited is identified by a track and sector location on the disk. Thedispenser is then moved to the appropriate track while the disksubstrate rotates. When the appropriate cell is positioned below thedispenser (as referenced by the appropriate sector on the track), adroplet of monomer solution is released.

Control of the droplet size may be accomplished by various techniques.For example, in one embodiment, a conventional micropipetting instrumentis adapted to dispense droplets of five nanoliters or smaller from acapillary. Such droplets fit within regions having diameters of 300 μmor legs when a non-wetting mask of the invention is employed.

In another embodiment, the dispenser is a piezoelectric pump thatgenerates charged droplets and guides them to the reaction region by anelectric field in a manner analogous to conventional ink-jet printers.In fact, some ink-jet printers can be used with minor modification bysimply substituting a monomer containing solution for ink. For example,Wong et al., European Patent Application 260 965, incorporated herein byreference for all purposes, describes the use of a commercial printer toapply an antibody to a solid matrix. In the process, a solutioncontaining the antibody is forced through a small bore nozzle that isvibrating in a manner that fragments the solution into discretedroplets. The droplets are subsequently charged by passing through anelectric field and then deflected onto the matrix material.

A conventional ink drop printer includes a reservoir in which ink isheld under pressure. The ink reservoir feeds a pipe which in connectedto a nozzle. An electromechanical transducer is employed to vibrate thenozzle at some suitable high frequency. The actual structure of thenozzle may have a number of different constructions, including a drawnglass tube which is vibrated by an external transducer, or a metal tubevibrated by an external transducer (e.g. a piezoelectric crystal) or amagnetostrictive metal tube which is magnotostrictively vibrated. Theink accordingly is ejected from the nozzle in a stream which shortlythereafter breaks into individual drops. An electrode may be presentnear the nozzle to impart a charge to the droplets. Conventional inkdrop dispensers are described in U.S. Pat. Nos. 3,281,860 and 4,121,222,which are incorporated by reference herein for all purposes.

In a different preferred embodiment, the reactant solutions aredelivered from a reservoir to the substrate by an electrophoretic pump.In this device, a thin capillary connects a reservoir of the reactantwith the nozzle of the dispenser. At both ends of the capillary,electrodes are present to provide a potential difference. As is known inthe art, the speed at which a chemical species travels in a potentialgradient of an electrophoretic medium is governed by a variety ofphysical properties, including the charge density, size, and shape ofthe species being transported, as well as the physical and chemicalproperties of the transport medium itself. Under the proper conditionsof potential gradient, capillary dimensions, and transport mediumrheology, a hydrodynamic flow will be set up within the capillary. Thus,in an electrophoretic pump of the present invention, bulk fluidcontaining the reactant of interest is pumped from a reservoir to thesubstrate. By adjusting the appropriate position of the substrate withrespect to the electrophoretic pump nozzle, the reactant solution isprecisely delivered to predefined reaction regions.

In one particularly useful application, the electrophoretic pump is usedto produce an array containing various fractions of an unknown reactantsolution. For example, an extract from a biological material such asleaf or a cell culture might contain various unknown materials,including receptors, ligands, alkaloids, nucleic acids, and evenbiological cells, some of which may have a desired activity. If areservoir of such extract in electrophoretically pumped, the variousspecies contained therein will move through the capillary at differentrates. Of course, the various components being pumped should have somecharge so that they can be separated. If the substrate is moved withrespect to the dispenser while the extract components are beingseparated electrophoretically, an array containing various independentspecies is produced. Thin array is then tested for activity in a bindingassay or other appropriate test. Those elements of the array that showpromising activity are correlated with a fraction of the extract whichis subsequently isolated from another source for further study. In someembodiments, the components in the extract solution are tagged with, forexample, a fluorescent label. Then, during the process of delivering thesolution with the electrophoretic pump, a fluorescence detectordetermines when labeled species are being deposited on the substrate. Insome embodiments, the tag selectively binds to certain types ofcompounds within the extract, and imparts a charge to those compounds.

Other suitable delivery means include osmotic pumps and cell(biological) sorters. An osmotic pump delivers a steady flow of solutionfor a relatively long period. The construction of such pumps iswell-known in the art, generally incorporating a solution of the extractof interest within a solvent permeable bag. Osmotic pressure is appliedto the extract solution by solvent molecules diffusing across the bag toequalize a concentration difference. The extract is thus forced out of anozzle in the bag at a constant rate. Cell sorters are also well-knownin the art, and can be used in applications where it is desirable toapply single biological cells to distinct locations on the substrate.

Although the above embodiments have been directed to systems employingliquid droplets, minuscule aliquots of each test substance can also bedelivered to the cell as miniature pellets. Such pellets can be formedfrom the compound of interest (e.g. ligands for use in an affinityassay) and one or more kinds of inert binding material. The compositionof such binders and methods for the preparation of the pellets will beapparent to those of skill in the art. Such “mini-pellets” will becompatible with a wide variety of test substances, stable for longperiods of time, suitable for easy withdrawal from the storage vesseland dispensing (i.e., non-tacky, preferably suspendable in a liquid suchas physiological buffer), and inert with respect to the binding activityof receptors.

In preferred embodiments, the reactant solutions in each predefinedregion are prevented from moving to adjacent regions by appropriatebarriers or constraining regions. For example to confine aqueous monomersolutions, a hydrophilic material in used to coat the reaction regions,while a hydrophobic material is used in preferred embodiments to coatthe region surrounding the individual reaction regions. Of course, whennon-aqueous or nonpolar solvents are employed, different surfacecoatings are generally preferred. By choosing appropriate materials(substrates, hydrophobic coatings, and reactant solvents), the contactangle between the droplet and the substrate in advantageouslycontrolled. Large contact angles between the reactant droplets and thesubstrate are desired because the solution then wets a relatively smallreaction region with shallow contact angles, on the other hand, thedroplet wets a larger area. In extreme cases, the droplet will spread tocover the entire surface.

The contact angle is determined by the following expression, known asYoung's equation:cos θ=(σ_(m)−σ_(d))/σ_(b)where θ is the wetting angle, σ_(m) is the solid-air tension, σ_(d) isthe solid-liquid tension, and σ_(b) is the liquid-air surface tension.The values of these surface tensions are governed by thermodynamicconsiderations including the chemical constituents of the liquid and thesolid substrate. The liquid-air surface tension for various chemicals iseasily measured by a variety of techniques such an those described inAdamson, Physical chemistry of Surfaces, John Wiley and Sons, 5th Ed.(1990) which is incorporated herein by reference for all purposes. Thedifference of the solid-liquid and solid-air tensions can, for a givensystem, be determined empirically from a Zisman plot. In thin approach,the contact angles are measured for a homologous series of liquids on agiven solid surface. For some liquid in the series, a “critical contactangle” is observed, beyond which lower surface tension liquids wet thesurface. The liquid-air surface tension of the liquid at this criticalcontact angle is assumed to be the surface tension of the solid. Thisapproach has been found to provide quite reasonable results for lowenergy solids such as Teflon, polyethylene, hydrocarbons, etc. Theinformation gained from such studies is used to optimize substratecompositions to increase wetting angles for given reactant solutions inthe array.

Methods for controlling chemical composition and therefore the localsurface free energy of a substrate surface include a variety oftechniques apparent to those skilled in the art. Chemical vapordeposition and other techniques applied in the fabrication of integratedcircuits can be applied to deposit highly uniform layers on selectedregions of a surface. As a specific example, the wettability of asilicon wafer surface has been manipulated on the micrometer scalethrough a combination of self-assembled monolayer depositions andmicromachining. See Abbott et al., “Manipulation of the Wettability ofSurfaces on the 0.1 to 1 Micrometer Scale Through micromachining andMolecular Self-Assembly” Science, 257 (Sep. 4, 1992) which isincorporated herein by reference for all purposes.

In a preferred embodiment, the perimeters of the individual regions areformed on a hydrophilic substrate defined by selectively removinghydrophobic protecting groups from the substrate surface. For example, amono-layer of hydrophobic photoprotecting groups can be coupled to, forexample, linker molecules attached to the substrate surface. The surfacethen is selectively irradiated (or, otherwise activated by, for example,acid) through a mask to expose those areas where the reaction regionsare to be located. This cleaves the protecting groups from the substratesurface, causing the reaction regions to be less hydrophobic than thesurrounding area. This process produces a high density of reactionregions on the substrate surface. Because hydrophobic materials havelower surface free energies (surface tensions) than water, the solutiondroplet in the cell beads rather than spreads.

In some preferred embodiments, the substrate is prepared by firstcovalently attaching a monolayer of the desired reactive functionalgroup (e.g. amine, hydroxyl, carboxyl, thio, etc.), which is protectedby a hydrophobic photolabile protecting moiety. If the substrateprovides a glass surface, the monolayer may be deposited by a silanationreaction as shown below.

In the above structures, Y in a spacer group such as a polymethylenechain, X in a reactive protected group such as NH, C(O)O, O, S, etc.,and Pr is a hydrophobic photolabile protecting group.

In an alternative preferred embodiment shown below, the substratesurface is first derivatized by, for example, a silanation reaction withappropriates to provide an amine layer. A molecule including a spacer, areactive group, and a photolabile group is then coupled to the surface.

The photolabile protecting group should be sufficiently hydrophobic anto render the substrate surface substantially non-wettable. Removal ofthe protecting group in specific areas by exposure to light through asuitable mask, liberates the reactive functional groups. Because thesegroups are hydrophilic in character, they will render the substratewettable in the exposed regions.

The class of nitrobenzyl protecting groups, which is exemplified by thenitroveratryl group, imparts significant hydrophobicity to glasssurfaces to which a member of the class is attached. The hydrophobicityof the basic nitrobenzyl protecting group is enhanced by appending groupchain hydrocarbon substituent. Exemplary hydrophobic chains includeC₁₂H₂₅ (lauryl) or C₁₈H₃₇ (stearyl) substituents. The syntheses ofsuitably activated forms (bromide, chloromethyl ether, and oxycarbonylchloride) of a typical protecting group is schematically outlined inFIG. 14.

The spacer group (“Y”) contributes to the net hydrophobic or hydrophilicnature of the surface. For example, those spacers consisting primarilyof hydrocarbon chains, such as —(CH₂)_(n)—, tend to decreasewettability. Spacers including polyoxyethylene (—(CH₂CH₂O)_(a) ), orpolyamide (—(CH₂CONH)_(a)) chains tend to make the surface morehydrophilic. An even greater effect is achieved by using spacer groupswhich possess, in addition to the protected functional group, several“masked” hydrophilic moieties. This is illustrated below.

In preferred embodiments, the hydrophilic reaction regions is atwo-dimensional circle or other shape having an aspect ratio near one(i.e. the length is not substantially larger or smaller than the width).However, in other embodiments, the hydrophilic region may take the formof a long channel which is used to direct flowing reactants in themanner described above.

In still other embodiments, the reaction regions are three-dimensionalareas defined by, for example, gaskets or dimples on the substratesurface. The dimples or gaskets may also act as identification marksdirecting the dispenser to the region of interest.

If the solvent (or other liquid used to deliver the reactant) has asufficiently high vapor pressure, evaporation can cause the reactantconcentration to increase. If left unchecked, this process ultimatelycauses the solute to precipitate from solution. The effects ofevaporation can be minimized by sealing selected regions of thesubstrate when they need not be accessible. Alternatively, the partialpressure of volatile reagents can be adjusted so that the liquid andvapor phase fugacities are equalized and the thermodynamic force drivingevaporation is reduced. The partial pressure of the reagents may beincreased by providing a relatively large reservoir of volatile reagentsin a sealed chamber. For example, solvents having a low vapor pressureunder the conditions of interest can be used. In some cases, evaporationcan be further controlled by application of a film or coverplate havinga reverse array pattern. Other methods of preventing evaporation arewell-known in the physical chemical arts and may be used in the presentinvention.

In some preferred embodiments, evaporation is advantageously employed toaccelerate hybridization of target oligonucleotides with immobilizedoligonucleotides in the reaction regions. In one specific embodiment,fluorescently tagged or otherwise labelled target oligonucleotides insolution (e.g., a solution containing a salt such as ammonium acetate ormagnesium chloride) are delivered to reaction regions containingimmobilized probe oligonucleotide. As the volatile salt solutionevaporates from the reactant droplet (in the same manner as solventevaporates from an ink droplet deposited by an ink jet printer), alocally high concentration ratio of target to probe oligonucleotideresults, accelerating hybridization. If hybridization is carried out atroom temperature, ten minutes to a few hours are typically required tocomplete the reaction. After sufficient time, the unhybridized DNA iswashed or otherwise removed from the substrate. Finally, the substrateis imaged to detect regions in which the probe and target DNA havehybridized. Of course, evaporation can be advantageously employed toincrease the local concentration of non-DNA solutes in a variety ofreactions besides hybridization. For example in some embodiments,receptor solutions are sufficiently volatile that the local receptorconcentration increases in the reaction regions containing peptides, forexample, to be screened.

The arrays produced according to the above spotting embodiments aregenerally used in much the same manner as the arrays produced by theflow channel embodiments described above. For example, the arrays can beused in screening with fluorescein labelled receptors as described inPCT Publication No. WO92/10092, previously incorporated by reference.

VI. Alternative Embodiments

According to some embodiments of the invention, microvalve structuresare used to form channels along selected flow paths on the substrate.According to these embodiments, an array of microvalves is formed andoperated by an overlying or underlying array of electrodes that is usedto energize selected valves to open and close such valves.

FIG. 15 illustrates such a structure, FIG. 15 a illustrating the systemin end view cross-section and FIG. 15 b illustrating the system in topview. The structure shown therein provides for only two synthesischambers for the purpose of clarity, but in most embodiments a fargreater number of chambers will be provided. Microvalves are discussedin detail in, for example, Zdeblick, U.S. Pat. No. 4,966,646, andKnutti, “Advanced Silicon Microstructures,” ASICT Conference (1989),both incorporated herein by reference for all purposes.

As shown therein, a substrate 602 is provided with a plurality ofchannels 604 formed using photolithographic, or other relatedtechniques. The channels lead up to a synthesis chamber 606. At the endof each channel is valve structure 608. As shown in FIG. 15, thechannels lead up to the chambers, but may be isolated from the chambersby the valves. Multiple valves may be provided for each chamber. In theparticular structure shown in FIG. 15, the right valve on the leftchamber and the left valve on the right chamber are open while theremaining valves are closed. Accordingly, if reagent is delivered to thetop of the substrate, it will flow through the open channel to andthrough the chamber on the left, but not the one on the right.Accordingly, coupling steps may be conducted on the chamber withselected reagents directed to selected chambers, using the techniquesdiscussed above.

According to some embodiments, a valve is supplied on one side of thechamber 606, but the valve on the opposite side is replaced by asemi-permeable membrane. According to these embodiments, it becomespossible to flow a selected reagent into the chamber 606 and,thereafter, flow another selected reagent through the flow channeladjacent the semi-permeable membrane. The semi-permeable membrane willpermit a portion of the material on one side or the other to passthrough the membrane. Such embodiments will be useful in, for example,cell studies.

Screening will be performed by, for example, separating or cutting twohalves of the device, enabling screening by, for example, contactingwith a fluorescein labelled antibody, or the like followed byphotodetection.

FIGS. 16 a and 16 b illustrate another alternative embodiment of theinvention which combines the mechanical polymer synthesis techniquesdisclosed herein with light-directed synthesis techniques. According tothese embodiments, a substrate 401 is irradiated in selected regions,shown an the stripes in FIG. 16 a. The surface of the substrate isprovided with photoremovable groups in accordance with PCT PublicationNo. WO92/10092 (previously incorporated by reference) on, for example,amine groups in the specific case of peptide synthesis. During this stepregions 701, 702, and 703 of the substrate, among others, aredeprotected, leaving remaining regions of the substrate protected byphotoremovable groups much as nitroveratryl oxycarbonyl (“NVOC”).According to a specific embodiment of the invention the widths of theirradiated regions equal the widths of the protected regions of thesubstrate.

Thereafter, as shown in FIG. 16 b the substrate is contacted with achannel block 407. In the particular embodiment shown in FIG. 16 b, thechannels 704, 705, and 707 are aligned with the regions 701, 702, and703, respectively, on the substrate 401. As will be apparent, specificembodiments of the invention provide for irradiated regions and channelsin the form of stripes, which are aligned during this step. Otherembodiments, however, will provide for other shapes of irradiatedregions and channels, and other relative orientations of the irradiatedregions and channels. The channel block and substrate will be alignedwith, for example, an alignment mark placed on both the substrate andthe channel block. The substrate may be placed on the channel blockwith, for example, a vacuum tip.

Thereafter, a selected reagent is flowed through or placed in thechannels in the channel block for coupling to the regions which havepreviously been exposed to light. As with the flow channel embodimentsdescribed above, the substrate may be placed in contact with a prefilledchannel block is some embodiments to avoid compression of the channelblock to the substrate and dead spots during pumping. According topreferred aspects of the invention, a different reagent flows througheach of the channels 701, 702, and 703 such as, for example, a reagentcontaining monomers A, B, and C. The process may then, optionally,involve a second coupling step in which the substrate is translated by,e.g., one channel width, to provide coupling of a monomer in the regionsbetween the original channels.

Thereafter, the process of directed irradiation by light, followed bycoupling with the channel block is repeated at the previously unexposedregions. The process is then preferably repeated again, with the stripesof the mask and the channel block rotated at, for example, 90 degrees.The coupling steps will provide for the formation of polymers havingdiverse monomer sequences at selected regions of the substrate throughappropriate translation of the mask and substrate, and throughappropriate mask selection. Through a combination of the light-directedtechniques and the mechanical flow channel techniques disclosed herein,greater efficiency in forming diverse sequences is achieved, becausemultiple monomers are coupled in a single irradiation/coupling step.

In light-directed methods, the light shown through the mask isdiffracted to varying degrees around the edges of the dark regions ofthe mask. Thus, some undesired removal of photosensitive protectinggroups at the edges of “dark” regions occurs. This effect is exacerbatedby the repeated mask translations and subsequent exposures, ultimatelyleading to inhomogeneous synthesis sites at the edges of the predefinedregions. The effect is, of course, dependent upon the thickness of theglass substrate and the angle at which the light is diffracted. If themask is positioned on the “backside” of the substrate, a diffractionangle of 2.5° and a substrate thickness of 0.7 mm creates a 60 μm stripof light (of variable intensity) flanking each edge. For a 0.1 mm thicksubstrate, the strip is approximately 5 μm wide.

To reduce these “bleed-over” effects of diffraction, a pinhole mask maybe employed to activate and/or define reaction regions of the substrate.Thus, for example, light shown through the pinhole mask is directed ontoa substrate containing photoremovable hydrophobic groups. The groups inthe illuminated regions are then removed to define hydrophilic reactionregions. In one specific embodiment, the pinhole mask contains a seriesof circular holes of defined diameter and separation, e.g., 20 μmdiameter holes spaced 50 μm apart. In some preferred embodiments, astationary pinhole mask is sandwiched between the substrate and atranslational mask of the type described in PCS Publication No.WO92/10092. In this manner selected regions of the substrate can beactivated for polymer synthesis without bleed-over. The translationalmask is used to illuminate selected holes of the stationary pinholemask, and is aligned such that its edges dissect the distance separatingthe holes of the stationary mask thereby eliminating diffractive removalof photopreotecting groups at neighboring sites. Because there isnegligible bleed-over incident light, inhomogeneous synthesis at sitesjuxtaposed along the edge is eliminated. The resulting circular sitesdo, of course, contain variable sequence density due to diffraction atthe edges of the pinhole mask, but the sequences at each predefinedregion are homogeneous. In addition, each synthesis region is surroundedby a “dark” region when the substrate is probed with a labeled target.Thus, no bleed-over fluorescence signal is introduced by binding atneighboring sites.

A pinhole mask containing 20 μm circular holes separated by 50 μmrequires a total synthesis area for the complete set of octanucleotidesof only 1.78 cm². For a given pinhole mask, thinner substrates allow forsmaller reaction sites separated by larger distances. However, the areafrom which reliable data can be obtained is also reduced when smallersites arm used. The density of reaction sites is ultimately determinedby the diffraction angle and the distance between the pinhole mask andthe reaction regions (typically the substrate thickness).

Although the discussion so far has focused upon circular pinholes, othershapes such as slots, squares, crescents, etc. may be employed as isappropriate for the selected delivery method. Thus, for some flowchannel embodiments, linear or serpentine slots may be desired.

In alternative preferred embodiments, the pinhole mask takes the form ofa layer coated on the substrate. This avoids the need for a separatestationary mask to generate the dot pattern. In addition, the surfacelayer provides well defined synthesis regions in which to depositreactants according to the spotting embodiments described above.Further, the surface pinhole mask is conveniently embossed with localreference coordinates for use in navigational systems used to delivermonomer solutions to proper regions as described above. Preferredpinhole masks are made from opaque or reflective materials such aschrome.

VI. Examples

A. Leak Testing

An initial experiment was conducted using a flow channel device toensure that solutions could be delivered to selected locations of asubstrate and be prevented from contacting other areas. Additionally,the experiment was used to demonstrate that reagents could be deliveredin a uniform manner.

Accordingly, a flat piece of conventional glass having dimensions ofabout 42 mm×42 mm was derivatized with aminopropyltriethoxysilane. Theentire slide was deprotected and washed using conventional techniques. Afluorescein marker of FITC was then injected into flow channels formedwhen a block of KeIF™ 81 with 10 channels of 1 mm depth and 1 mm widthwere brought into contact with the substrate. The fluorescein marker wasin a solution of DMF and flowed through the channels by injecting thematerial into the groove with a manual pipet.

Fluorescein dye was similarly injected into every other channel in theblock, the block was rotated, and the process was repeated. The expectedresulting plot of fluorescent intensity versus location is schematicallyillustrated in FIG. 17. Dark regions are shown at the intersections ofthe vertical and horizontal stripes, while lighter grey atnon-intersecting regions of the stripes. The dark grey regions indicateexpected regions of high dye concentration, while the light regionsindicate regions of expected lower dye concentration.

FIG. 18 is a mapping of fluorescence intensity of a portion of an actualslide, with intensity data gathered according to the methods of PCTPublication No. WO92/10092, previously incorporated by reference. Theresults agree closely with the expected results, exhibiting highfluorescence intensity at the intersection of the channels (about 50%higher than non-intersecting regions of the stripes), and lowerfluorescence intensity at other regions of the channels. Regions whichwere not exposed to fluorescence dye show little activity, indicating agood signal-to-noise ratio. Intersections have fluorescence intensityabout 9× as high as background. Also, regions within the channels showlow variation in fluorescence intensity, indicating that the regions arebeing evenly treated within the channels.

B. Formation of YGGFL (SEQ ID NO:1)

The system was used to synthesize four distinct peptides: YGGFL (SEQ IDNO:1), YpGFL (SEQ ID NO:2), pGGFL (SEQ ID NO:3), and ppGFL (SEQ ID NO:6)(the abbreviations are included in Stryer, Biochemistry, Third Ed.(1988), previously incorporated herein by reference; lower case lettersindicate D-optical isomers and upper case letters indicate L-opticalisomers). An entire glass substrate was derivatized with TBOC-protectedaminopropyltriethoxysilane, deprotected with TFA, coated withFMOC-protected caproic acid (a linker), deprotected with piperidine, andcoated with FMOC-protected Glycine-Phenylalanine-Leucine (GFL).

This FMOC-GFL-coated slide was sealed to the channel block, and all 10grooves were deprotected with piperidine in DMF. After washing thegrooves, FMOC Glycine (G) was injected in the odd grooves, and FMOCd-Proline (p) was injected in the even grooves. After a two-hourcoupling time, using standard coupling chemistry, all grooves werewashed with DMF. The grooves were vacuum dried, the block removed androtated 90 degrees. After resealing, all grooves were deprotected withpiperidine in DMF and washed. FMOC Tyrosine (Y) was injected in the oddgrooves, and FMOC p in the even grooves. After coupling the grooves werewashed and vacuum dried. Accordingly, 25 regions of each of thecompounds YGGFL (SEQ ID NO:1), YpGFL (SEQ ID NO:2), pGGFL (SEQ ID NO:3),and ppGFL (SEQ ID NO:6) were synthesized on the substrate. The substratewas removed and stained with FITC-labelled antibodies (Herz antibody3E7).

A section of the resulting slide illustrating fluorescence intensity isshown in FIG. 19. White squares are in locations of YGGFL (SEQ ID NO:1).The darkest regions are pGGFL (SEQ ID NO:3) and ppGFL (SEQ ID NO:6). TheYGGFL (SEQ ID NO:1) sites were the most intense, followed by the YpGFL(SEQ ID NO:2) sites. The PGGFL (SEQ ID NO:3) and ppGFL (SEQ ID NO:6)intensities were near background levels, consistent with expectedresults with the Herz antibody.

Quantitative analysis of the results show overall intensity ratios forYGGFL (SEQ ID NO:1):YpGFL (SEQ ID NO:2):pGGFL (SEQ ID NO:3):ppGFL (SEQID NO:6) as 1.7:1.5:1.1:1.0. However, since there is a large standarddeviation on the YGGFL (SEQ ID NO:1) and YpGFL (SEQ ID NO:2), comparingall the sites with each other may not accurately represent the actualcontrasts. Comparing the intensities of sites within the same “stripe”gives larger contrasts, although they remain on the order of 2:1.

C. 100 Micron Channel Block

A grid pattern of fluorescein isothiocyanate coupled to a substrate wasmade by using a flow cell of this invention. A two by three inchNVOC-derivatized substrate was photolyzed through a mask to produce 400micron activated bands on one axis. An etched silicon channel blockhaving 64 parallel 100 micron channels separated by 100 micron walls wasthen clamped to the substrate on the other axis (i.e., perpendicular tothe axis of 400 micron activated bands). The clamping assemblyconsisting of aluminum top and bottom clamp plates was used. Pressurewas applied by tightening two bolts with a torque wrench to 400 psi. A 7mM fluorescein isothiocyanate solution was flowed through the channelsby pipetting directly to exposed channel ends.

An image of the substrate (FIG. 20) showed regions of high fluorescenceindicating that the fluorescein had bound to the substrate. Whitesquares indicating fluorescein binding were present as 400 micronhorizontal stripes on the photolyzed regions within the 100 micronvertical flow paths. Contrast ratios of 8:1 were observed between thechannels and the channel spacings. This demonstrates the nearly completephysical isolation of fluid passing through 100 micron channels under400 psi of clamping pressure.

D. Channel Matrix Hybridization Assay

A center region of a two by three inch slide was derivatized withbis(2-Hydroxyethyl)aminopropyltriethoxy silane. Six nucleosides werethen coupled to the entire reaction region using a synthesis processconsisting of deprotection, coupling, and oxidation steps for eachmonomer applied. These first six nucleosides were coupled in a reactionregion defined by a 0.84 inch diameter circular well in an aluminumtemplate clamped to the two by three inch slide.

The seventh and eighth monomers were applied to the substrate by flowingmonomer solutions through 100 micron channels in an etched siliconchannel block (employed in Example C above). The seventh base wascoupled along the long axis (vertical) of the two-inch by three-inchslide, and the eighth base perpendicular to the seventh, along the shortaxis (horizontal) of the slide. This defined an active matrix region of1.28 by 1.28 cm having a density of 2,500 reaction regions per squarecentimeter.

The channel block was centered over the reaction region and clamped tothe substrate using a clamping assembly consisting of machined aluminumplates. This aligned the two inch by three inch substrate relative tothe channel block is the desired orientation. Rotation of the top clampplate and channel block relative to the bottom clamp plate and substratebetween the seventh and eighth coupling steps provided for the matrix ofintersecting rows and columns.

In the top clamp plate, fluid delivery wells were connected tolaser-drilled holes which entered individual channels from the backsurface of the channel block. These delivery wells were used to pipettecoupling reagents into channels while the channel block was clamped tothe substrate. Corresponding fluid-retrieval wells were connected tovacuum at the downstream of the channel block, drawing fluid through thechannels and out to a waste container. Thus continuous fluid flow everthe substrate in the channel region during coupling steps was achieved.

The complete octamer synthesized at the channel intersections formed bythe seventh and eighth coupling steps had the following sequences:Substrate—(3′) CGCAGCCG (5′) (SEQ ID NO:4).

After completion of the synthesis process, cleavage of exocyclic amineswas performed by immersion of the reaction region in concentratedammonium hydroxide. The reaction region was then incubated at 15° C. forone hour in a 10 nM solution of the complementary base sequence 5′GCGTCGGC-F (SEQ ID NO:5), where “F” is a fluorescein molecule coupled tothe 3′ end of the oligonucleotide. The target chain solution was thenflushed from the reaction region and replaced with neat 6× SSPE buffer,also at 15° C. Finally, the reaction region was then scanned using alaser fluorescence detection system while immersed in the buffer.

The brightest regions in the resulting image (FIG. 21) correspond tochannel intersections where a full octamer was synthesized on thesubstrate surface. Vertical columns on the image displayed the channelregions where the seventh base was coupled, while horizontal rowsdisplay the channel regions where the eighth base was coupled.Brightness in the channel intersection regions indicated hybridizationbetween the fluoresceinated target chain and the complementary chainsynthesized and bound to the substrate in these regions. The verticalstripes of the image showed a consistent brightness with regions ofsignificantly greater brightness at the intersection regions. Thehorizontal stripes did not contain the consistent brightness of thevertical stripes, but did have regions of brightness at theintersections with the vertical stripes.

The consistent brightness along the seventh monomer axis (vertical)indicated partial hybridization of the target chain in areas where sevenof the eight complementary bases were coupled to the substrate surface.The lack of brightness along the eighth monomer axis (horizontal) isconsistent with the expectation that a chain of six matching bases boundto the substrate surface will not hybridize effectively to an octamer insolution (heptamers with six matching bases followed by a mismatch atthe seventh position). The darker background consists of hexamersconsisting of the first six monomers coupled to the entire reactionregion.

FIG. 22 is a magnified view of the image in FIG. 21. FIG. 22demonstrates that the separate reaction regions are well resolved.

VII. Conclusion

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example avariety of substrates, receptors, ligands, and other materials may beused without departing from the scope of the invention. The scope of theinvention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

1. An automated method of forming an array of ligands on a supporthaving localized areas comprising (a) locating a dispenser to dispense asolution a distance away from a surface of the support; (b) dispensing afirst monomer in a volume of solution in a single coupling step of lessthan 5 nl onto the surface of the support to occupy a localized areasmaller than 1 cm²; (c) allowing the first monomer to attach directly orindirectly to the surface of the support at the localized area; (d)repeatedly dispensing an additional group of added monomers in a volumeof solution in a single coupling step of less than 5 nl at a same ordifferent localized area in a manner to couple with a compound at one ormore localized areas until an array of at least 100 different ligands,each at an individual localized area is formed and wherein density oflocalized areas on the support is at least about 1000 localized areasper cm² of surface of the support.
 2. The method of claim 1 wherein themonomer is dissolved in solution.
 3. The method of claim 1 wherein themonomer is in the form of a pellet.
 4. The method of claim 1 wherein thesupport further comprises a cover plate.
 5. The method of claim 1wherein the step of dispensing includes a dispenser positioned betweenabout 5 microns and about 50 microns away from the support.
 6. Themethod of claim 1 wherein the step of dispensing includes a dispenserpositioned about 10 microns away from the support.
 7. The method ofclaim 1 wherein the volume fits within a region having a diameter ofless than about 300 microns.
 8. The method of claim 1 wherein themonomer comprises a nucleotide or an amino acid.
 9. The method of claim1 wherein the ligand comprises a nucleic acid, oligonucleotide,polynucleotide, peptide, or polypeptide.
 10. The method of claim 1wherein the ligand comprises at least 2 monomers.
 11. The method ofclaim 1 wherein the ligand comprises greater than 100 monomers.
 12. Themethod of claim 1 wherein the ligand comprises 2, 3, 4, 5, 6, 10, 15,20, 30, 40, 50, 75, or 100 monomers.
 13. The method of claim 1 whereinthe support is selected from the group consisting of substantially flatsubstrates, substrates having raised or depressed regions, beads, gels,sheets, particles, strands, precipitates, spheres, containers,capillaries, pads, slices, films, plates, and slides.
 14. The method ofclaim 1 wherein the support comprises a gel.
 15. The method of claim 1wherein the support comprises biological materials, nonbiologicalmaterials, organic materials or inorganic materials.
 16. The method ofclaim 1 wherein the support is a disc, square, or circle.
 17. The methodof claim 1 wherein the one or more localized areas are smaller than 1mm².
 18. The method of claim 1 wherein the one or more localized areasare smaller than 0.5 mm².
 19. The method of claim 1 wherein the one ormore localized areas are smaller than 10,000 μm².
 20. The method ofclaim 1 wherein the one or more localized areas are smaller than 100μm².
 21. The method of claim 1 wherein an array of at least 1000different ligands at different localized areas is formed.
 22. The methodof claim 1 wherein an array of at least 10,000 different ligands atdifferent localized areas is formed.
 23. The method of claim 1 whereinan array of at least 100,000 different ligands at different localizedareas is formed.
 24. The method of claim 1 wherein an array of at least1,000,000 different ligands at different localized areas is formed. 25.The method of claim 1, wherein an array of at least 1000 differentpolymers occupying localized areas within 1 cm² of the surface of thesupport.
 26. The method of claim 1, wherein the support comprises glass,derivatized glass, pyrex, quartz, a polymeric material, polystyrene,polycarbonate, silicon or a gel.
 27. The method of claim 2, wherein thesolution comprises an aqueous solution.
 28. The method of claim 1wherein the step of dispensing includes a dispenser comprising aplurality of dispensing units, wherein the plurality of dispensing unitsis in fluid communication with a solution comprising a monomer andwherein steps (a) and (c) comprise dispensing a volume of solution in asingle coupling step of less than 5 nl from one or more of the pluralityof dispensing units.
 29. The method of claim 1, wherein the supportbears at least two reference points for positioning the dispenser overat least one of said localized areas for dispensing of the volume ofsolution.
 30. The method of claim 29, wherein the reference pointscomprise global reference points for positioning the dispenser over alocal region of the surface of the support, and local reference pointswithin the local region for positioning the dispenser over a localizedarea within the local region.
 31. The method of claim 29, wherein thedispenser further comprises a camera for identifying the referencepoints.
 32. The method of claim 29 further comprising the step ofsensing changes in capacitance to identify the reference points.
 33. Themethod of claim 29 further comprising the step of sensing changes inlight intensity to identify the reference points.
 34. The method ofclaim 29 further comprising the step of sensing changes in resistivityto identify the reference points.
 35. The method of claim 29 furthercomprising the step of sensing changes in optical properties to identifythe reference points.
 36. The method of claim 29 further comprising thestep of sensing changes in magnetic properties to identify the referencepoints.
 37. The method of claim 28 wherein the plurality of dispensingunits comprises a manifold of delivery lines.
 38. The method of claim 28wherein the plurality of dispensing units comprises an array ofpipettes.
 39. The method of claim 28 wherein the plurality of dispensingunits comprises a series of tubes.
 40. The method of claim 28 whereinthe plurality of dispensing units includes control valves.
 41. Themethod of claim 1 wherein the monomer is bound indirectly to the surfaceof the support via a linker molecule.
 42. The method of claim 1 whereinthe step of dispensing includes a dispenser that is moved relative tothe support.
 43. The method of claim 1 wherein the support is movedrelative to a dispenser.
 44. The method of claim 1 wherein the one ormore localized areas are spaced less than about 3 mm apart.
 45. Themethod of claim 1 wherein the one or more localized areas are spacedless than between about 5 microns and 100 microns apart.
 46. The methodof claim 1 wherein the one or more localized areas has an angularrelation between each localized area of about 1 degree.
 47. The methodof claim 1 wherein the one or more localized areas has an angularrelation between each localized area of about 0.1 degree.
 48. The methodof claim 1 wherein the support comprises at least about 100 localizedareas.
 49. The method of claim 1 wherein the support comprises at leastabout 1000 localized areas.
 50. The method of claim 1 wherein thesupport comprises at least about 10,000 localized areas.
 51. The methodof claim 1 wherein the support comprises at least about 10,000 localizedareas per cm² of surface of substrate.
 52. The method of claim 1 whereinthe support comprises a strand including one or more of glass,derivatized glass, quartz, or a polymeric material.
 53. The method ofclaim 1 wherein the step of dispensing includes a dispenser comprising adispenser tip and a sheath encircling the dispenser tip and rigidlyextending a fixed distance beyond the dispenser tip.
 54. The method ofclaim 1 wherein the surface of the support comprises a hydrophilicsubstance.
 55. The method of claim 1 wherein the surface of the supportcomprises a hydrophobic substance.
 56. The method of claim 1 wherein thesurface of the support comprises a hydrophilic substance and ahydrophobic substance.
 57. The method of claim 1 wherein the surface ofthe support comprises a hydrophilic group.
 58. The method of claim 1wherein the surface of the support comprises a hydrophobic group. 59.The method of claim 1 wherein the surface of the support comprises ahydrophilic group and a hydrophobic group.
 60. The method of claim 1wherein the surface of the support comprises a photoresist.
 61. Themethod of claim 1 wherein the surface of the support is cleaned prior tothe step of dispensing the volume of solution.
 62. The method of claim 1wherein the step of dispensing includes a dispenser comprising apipette.
 63. The method of claim 1 wherein the step of dispensingincludes a dispenser comprising a capillary tube.
 64. The method ofclaim 1 wherein the step of dispensing includes a dispenser comprisingan electrophoretic pump.
 65. The method of claim 1 wherein the step ofdispensing includes a dispenser comprising an osmotic pump.
 66. Themethod of claim 1 wherein the step of dispensing includes a dispensercomprising a cell sorter.
 67. A method of forming an array of ligands ona support having localized areas comprising (a) locating a dispensercomprising a plurality of dispensing units a distance away from asurface of the support, wherein the plurality of dispensing units is influid communication with a solution comprising a monomer; (b) dispensinga volume of solution in a single coupling step of less than 5 nl fromthe dispenser, with the volume contacting the surface at a localizedarea smaller than 1 cm²; (c) allowing the monomer to attach directly orindirectly to the surface of the support at the localized area; (d)repeating steps a through c to attach a same or different monomer at asame or different localized area until an array of at least 100different ligands, each at an individual localized area is formed andwherein density of localized areas on the support is at least about 1000localized areas per cm² of surface of the support.
 68. The method ofclaim 67 wherein an array of at least 1000 different ligands atdifferent localized areas is formed.
 69. The method of claim 67 whereinan array of at least 10,000 different ligands at different localizedareas is formed.
 70. The method of claim 67 wherein an array of at least100,000 different ligands at different localized areas is formed. 71.The method of claim 67 wherein an array of at least 1,000,000 differentligands at different localized areas is formed.
 72. The method of claim67 wherein the monomer is an amino acid or a nucleotide.
 73. The methodof claim 67 wherein the ligand is a polypeptide or a nucleic acid.